1. Field of the Invention
This invention relates to immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for immunoassay in which enhancement of a detectable signal is achieved by modulation of chemical catalysis of an indicator reaction.
2. Description of the Invention
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous or homogeneous. Heterogeneous assays require a separation of bound tracer from free (unbound) tracer. Homogeneous assays do not require a separation step and thereby provide significant advantages in speed, convenience and ease of automation over heterogeneous assays.
Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing or large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must he followed.
Fluoroimmunoassay (FIA) uses fluorochromes as labels, provides direct detection of the label, and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
Enzymes have also been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. Detection of the signal with the naked eye is limited because the average individual can detect the presence of chromophores only down to about 10.sup.-5 or 10.sup.-6 M, and ligands to be detected or measured in biological fluids are often present in the range of 10.sup.-9 to 10.sup.-12 M.
EIA sensitivity can often be increased by spectrophotometric techniques; however, these procedures require expensive equipment. In another approach, the sensitivity is increased by cascade amplification. In this procedure, the number of detectable (generally colored) molecules is increased by use of two or more enzymes or enzyme derivatives in which a first enzyme conjugated to an assay ligand activates a second enzyme or enzyme derivative which catalyzes a color producing reaction or formation of a third enzyme. Exemplary of this technique is U.S. Pat. No. 4,463,090 to Harris.
U.S. Pat. No. 4,160,645 to Ullman discloses an immunoassay procedure in which there is measured the rate of a reaction between two redox reagents. A non-enzymic catalyst for the reaction, which may include a metal ion complex, is conjugated to a ligand. When the ligand binds to an antiligand, the approach of the redox reagents to the catalyst is inhibited and the rate of the reaction is modulated.
In U.S. Pat. No. 4,375,972 to Forgione et al. and Japanese patent No. JP/20133, immunoassays involving chemiluminescent reactions catalyzed by metal ions and metalloporphyrins conjugated to a ligand are disclosed.
Catalysis of chemical reactions by metal ions is well known and provides a means for analysis of metal ions present in trace amounts. These procedures employ the metal ion to catalyze a reaction which either produces a color or degrades an existing color, and generally depend on a linear relationship between the initial rate of reaction and the concentration of metal ion.
Trace metal analysis often employs organic compounds which alone have no catalytic activity, but which can affect the catalytic activity of the metal ion and thereby either increase or decrease the rate of the reaction. Such compounds are thus modulators of metal catalysis, and may be either activators or inhibitors. Use of modulators in trace metal analysis in water is discussed by Bontchev in Talanta 19, 675 (1972). The determination of the modulators themselves is discussed by Milovanovic in Microchem. J., 28, 437 (1983) and specific examples have been described by Antonov et al. and Dolmanova et al. (J. Anal. Chem. USSR, 31, 168 (1976) and J. Anal. Chem. USSR, 32 638 (1977)).
There is a need for a method of high sensitivity to detect ligands present in biological fluids at very low levels which does not require expensive instrumentation for signal detection. It is toward the fulfillment of this need that the present invention is directed.